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Molecular Biology

Molecular Biology. The RNA. Intended learning outcomes. *Describe the structures of mature mRNAs, tRNAs, rRNAs and their precursors *Outline the process of transcription *Explain how chromatin structure, transcription factors and microRNAs can regulate transcription

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Molecular Biology

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  1. Molecular Biology The RNA

  2. Intended learning outcomes *Describe the structures of mature mRNAs, tRNAs, rRNAs and their precursors *Outline the process of transcription *Explain how chromatin structure, transcription factors and microRNAs can regulate transcription *Describe the ribosome, rRNA and tRNAs and their functions in translation *Define the codon, anticodon and open reading frame *Describe initiation, elongation and termination of translation *Outline post synthetic sorting of proteins

  3. What are RNAs? • RNAs are molecules that, because of their chemical structure (extra hydroxyl group), can form complex molecules that can catalyse reactions and serve as intermediates between DNA and protein. • Instead of DNA's double helix, RNA is a single strand, which allows it to fold and bind to itself to form new structures and shapes (e.g. the clover leaf of tRNAs). • As RNAs are short lived and are not used to do store info (like the DNA hard drive) we can use slightly different chemicals in RNA, the uracil.

  4. The RNAs • RNA serves as templates (mRNA) • Part of machines (the rRNAs in ribosomes) • tRNAs that serves in the translation • RNAs can also regulate processes – e.g. the microRNAs that control inhibit translation • tRNAs, rRNAs and microRNAs are non-coding RNAs as they are not used to make (encode) protein. mRNAs are coding RNAs.

  5. RNA polymerases Eukaryotes contain three DNA dependent RNA polymerases • RNAPI synthesizes rRNA • RNAPII synthesizes mRNA • RNAPIII synthesizes tRNAs • RNA polymerases do not need a primer

  6. There are 3 stages to transcription. Initiation Elongation Termination Initiationis the formation of the basal transcription complex RNA polymerase II + basal transcription factors that assemble sequentially at the promoter. Promoter could be 100 bp or more!

  7. The transcription start site is called the promoter…the light switch! The first step in transcription is the binding of RNAP II to a DNA sequence in a particular region called the promoter

  8. Initiation of transcription • Following the binding of RNAP II to the promoter, a conformational change occurs such that the DNA double strand is unwound, and RNAP II is positioned at the start site. • This enzyme-DNA complex is called the open promoter complex. • RNAP II contains initiation and elongnation binding sites. • Elongation starts when the first complementary nucleotide binds to the elongation site of the RNAP II

  9. start Pre-initiation complex start start

  10. Initiation • RNA polymerase requires the presence of a core promoter sequence in the DNA. Core promoters are sequences within the promoter which are essential for transcription initiation. • The most common type of core promoter in eukaryotes is a short DNA sequence known as a TATA box, found -30 base pairs from the start site of transcription. • The TATA box, as a core promoter, is the binding site for a transcription factor called Transcription Factor II D (TFIID). • After TFIID binds to the TATA box, five more transcription factors (IIB, IIF, IIE, IIH, and helicase) and RNA polymerase combine around the TATA box in a series of stages to form a preinitiation complex. • One transcription factor has helicase activity and so is involved in the separating of opposing strands of double-stranded DNA to provide access to a single-stranded DNA template.

  11. Elongation • One strand of DNA, the template strand (or non-coding strand or nonsense), is used as a template for RNA synthesis. • As transcription proceeds, RNA polymerase uses base pairing complementarity with the DNA template to create an RNA copy. • Transcription is occurring 5' → 3'. • This produces an RNA molecule from 5' → 3', an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).

  12. Transcription Elongation DNA-RNA duplex is anti-parallel The initial RNA product synthesized by RNA Pol II is called a Primary transcript. The Primary transcriptundergoes several processing steps including Capping, Polyadenylation, and Splicingbefore a functional mRNA (messenger RNA) is produced

  13. 5’ End capping • The process of 5' capping is vital to creating mature messenger RNA, which is then able to undergo translation. Capping ensures the messenger RNA's stability while it undergoes translation. • The 5' cap is found on the 5' end of an mRNA molecule and consists of a guanine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage. This guanosine is methylated on the 7 position directly after capping.

  14. 5’ End capping • 7-methyl guanosine nucleotide cap added * One of the terminal phosphate groups is removed, leaving two terminal phosphates. * GTP is added to the terminal phosphates, losing two phosphate groups (from the GTP) in the process. This results in the 5' to 5' triphosphate linkage. * The 7-Nitrogen of guanine is methylated.

  15. Termination 3’-End polyadenylation (tailing)‏ • RNA endonuclease recognises the sequence(AAUAAA). • Cleaved 10-30 bases beyond AAUAAA. • adenylate polymerase adds 100-300 AMP residues.

  16. 5’ cap and poly A - functions • Regulation of nuclear export. • Prevention of degradation by exonucleases. • Promotion of 5' proximal intron excision. • Promotion of translation. The tail is shortened over time and when it is short enough, the mRNA is enzymatically degraded.

  17. Splicing • mRNA precursors (primary transcript) = exons and introns • Feature of eukaryotes but not prokaryotes • Splicing removes introns • Splice sites on modified pre-mRNA recognised by spliceosome http://uk.youtube.com/watch?v=FVuAwBGw_pQ

  18. Alternative splicing

  19. Transcription factors • DNA-binding factors that alter the transcriptional activity - recognize major and minor grooves of DNA helix. • TFs block transcription - repressors • TFs activate transcription - activators • Repressors bind control elements called silencers • Activators bind control elements called enhancers Euchromatin - active transcription Heterochromatin - genes are silent http://www.youtube.com/watch?v=_-9pROnSD-A

  20. Structure of Eukaryotic Promoters Control elements

  21. RNA Translation

  22. Protein Synthesis - Apparatus • Ribosomes: large and small ribosomal subunit • Complexes of RNA and protein • Eukaryoticribosome (80S) composed of one large (60S) and one small (40S) subunit • Prokaryoticribosome (70S) composed of one large (50S) and one small (30S) subunit S=Svedberg unit which relates to mass/shape/density

  23. Transcription site rRNA genes is in nucleolus - RNA pol I • Ribosomal proteins assemble with rRNAs in nucleolus • 45s rRNA precursor cleaved to 18, 6 and 28S rRNAs before leaving the nucleus • Ribosomes & mRNA assemble during protein synthesis

  24. tRNA is adapter molecule with anticodons that match codons in mRNA tRNA has Stem loop structure • aa covalently attached to 3' OH of CCA by aminoacyl tRNA synthetase • 20 aa tRNA synthetases • Each aa has set of tRNAs except UAA, UAG &UGA

  25. http://pubs.acs.org/cen/multimedia/85/ribosome/translation_bacterial.htmlhttp://pubs.acs.org/cen/multimedia/85/ribosome/translation_bacterial.html

  26. Codons • A codon is a trinucleotide sequence in mRNA read 5’ to 3’ • 20 amino acids in protein. • Surplus of codons. Degenerate sequence - several codons for each aa • Low frequency aa’s have one codon (tryptophan and methionine). • Start codon is ATG which gives methionine.

  27. Geneticcode

  28. Reading frames • A sequence of mRNA has 3 potential reading frames. • Only one reading frame used, selected by AUG start codon. • Stop codons terminate translation. • Start and stop codons define an ORF (Open reading frame).

  29. Translation - summary • The mRNA carries genetic information from the chromosomes to the ribosomes. • mRNA is read in a sequence of nucleotide triplets called codons. • The ribosome molecules translate this code to a specific sequence of amino acids by the aid of tRNA. • tRNAs are small noncoding RNA chains (74-93 nucleotides) that transport amino acids to the ribosome. • tRNAs have a site for amino acid attachment, and a site called an anticodon. The anticodon is an RNA triplet complementary to the mRNA triplet that codes for their cargo amino acid. • Aminoacyl tRNA synthetase catalyzes the bonding between specific tRNAs and the amino acids that their anticodons sequences call for.

  30. A polyribosome

  31. Mutations • Missense & Nonsense • Small deletions • Small insertions • Splicing • Regulatory

  32. …GGA GGT CAA… …Gly Gly Gln… …GGA GAT CAA… …Gly Asp Gln… Missense • The wrong sense The codon for one AA is changed to code for a different AA • May cause change in protein function or property

  33. Nonsense • Makes no sense • Change of a codon for an AA to a STOP codon • Truncated (shortened) protein …CTT GGA GAA GGT… …Leu Gly Glu Gly… 1480 AA protein …CTT TGA GAA GGT… …Leu STOP!!! 541 AA protein

  34. The big dog and the cat ate the rat and the cat Insertions & deletions of multiples of three letters retain the frame and make some kind of sense The dog ate the rat g and the ca The dot ate the rat g Insertions & deletions which are not a multiple of 3 destroy the reading frame > RUBBISH!! The doa ndt hec ata tet her at The dog agg ndt hec ata tet her at Reading frame The dog and the cat ate the rat

  35. … TTC TTT GTG GTG TTT TTA TCT GTG … … Phe Phe Val Val Phe Leu Ser Val … T … TTC TTG TGG TGT TTT TAT CTG TGC … … Phe Phe Trp Cys Phe Tyr Leu Cys … Small deletions 1 Coding sequence Out-of-frame • One or a few bases are deleted • Frameshift when not a multiple of 3 bases

  36. Small insertions (coding sequence) • Effects are similar to small deletions • If multiple of 3 bases, then one or more extra AAs will be encoded • If not a multiple of 3 bases, there will be a frameshift,

  37. Truncating micro-lesions • Nonsense mutations • The majority of frameshiftinginsertions, deletions and indels lead to large decreases in protein length • Almost always pathogenic due to loss of large amount of the normal protein Of 64 possible codons, 3 are termination signals Thus, on average, in random sequence 1 out of every 21 codons will be a stop codon

  38. Normal mRNA Missing exon Splicing mutations • Affect the splice donor, splice acceptor or splice regulatory sites • Exons may be skipped erroneously (or parts of intron sequences may be incorporated as exon) Often truncating – loss of sequence + possibility remaining sequence is out-of-frame

  39. GU AG AG GU AG 9 10 11 GU AA AG GU AG 9 10 11 GC 18kb GU AG 19 20 GU AG GU AG 19 20 Mutation creates new splice donor site which activates use of “cryptic” splice acceptor site > additional 84bp with in-frame STOP Examples of mutations affecting splicing

  40. Regulatory mutations • Mutations in promoter or enhancer sequences • May lead to upregulation or to downregulation of transcription • Either of these situations may be detrimental

  41. Protein sorting. Getting proteins to the right place The average mammalian cell contains 10,000 proteins. These proteins must be sorted to the correct membrane or aqueous compartment. This process is called protein targeting or sorting

  42. Signal sequences in polypeptides • Often short sequences near the N or C terminus • Signals recognised by receptors on organelles • Signals can activate enzymes • Used signal sequences at N-terminus can be removed with hydrolases to reveal successive signals • Signals that must be read several times are not cleaved

  43. Two sites of protein synthesis in the cell • First sorting decision - secretory pathway or cytosol • All ribosomes initiate protein synthesis in the cytoplasm and remain there or are directed to the ER. Proteins that carry a signalpeptide for the ER follow the secretory pathway

  44. Post-translational modification of proteins • Modifications to biologically active forms. Occur mainly in ER and Golgi complex • Glycosylation • Phosphorylation • Proteolytic processing • Disulphide bond formation

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